3.1 Species and geographical distribution of Impatiens plant resources in Eastern Yunnan
East Yunnan was located in the southwest of China, and had a relative altitude difference of about 4000m, with the highest altitude of 4040m and the lowest altitude of 107m. This region was influenced by both high and low latitudes and had a three-dimensional climate, which provided unique conditions for the growth and development of Impatiens. The 32 species of Impatiens collected this time were mainly concentrated in the high-humidity areas with an altitude of 1600 ~ 3000m, of which 2 species (I. dichroa and I. napoensis) were distributed at an altitude of 1300 ~ 1600m, accounting for about 6.25%; of which 9 species (I. rubrostriata, I. mengtzeana, I. linearisepala, etc.) were distributed at an altitude of 1600 ~ 2000m, accounting for about 28.125%; of which 20 species (I. racemosa, I. pinfanensis, I. desmantha, etc.) distributed at an altitude of 2000 ~ 3000m, accounting for about 62.5%; of which only I. rubrostriata was distributed above 3000m, accounting for about 3.125%.
From the perspective of the lateral distribution of species and the number of individuals, Kunming, Zhaotong and
Wenshan were the main distribution centers of Impatiens. Among them, 10 species of Impatiens were gathered in Zhaotong and Qujing regions, 10 species were collected in Wenshan and Honghe areas, and 12 species collected in Kunming and surrounding areas. The 32 Impatiens resources collected this time accounted for 70% of the total in eastern Yunnan. This study also found that I. siculifer, I. racemosa, and I. radiata, which are widely distributed laterally, had certain differences in their growth traits in the same ecological type. (The results are shown in Table 1)
3.2 Analysis of flower morphological characteristics of Impatiens in Eastern Yunnan
The genus Impatiens in eastern Yunnan had peculiar flower shapes and a wide range of vibrant colors. Among them, 15 species of yellow series account for 46.88%, 8 species of red series account for 25.00%, 7 species of white series account for 21.88%, and 2 species of purple series account for 6.25% of the total. In this investigation, it was found that the flower organs of the genus Impatiens had heterochromatic spots or stripes. The anatomical observation of flower morphology found that there were 2 or 4 sepals in the genus Impatiens, and most of the Impatiens with 2 sepals, I. linearisepala and I. corchorifolia has 4 sepals. The shape of standard shape of the genus Impatiens in eastern Yunnan was typically circular or nearly circular. Its wing lobes were all two-lobed, though some were three-lobed or more, and the base lobes were oval, nearly oval, nearly round, round, etc; the upper lobes were ax-shaped, broad ax-shaped, narrow ax-shaped, nearly elliptical, and half-moon-shaped; the shape of the lip shape was sac-shaped, funnel-shaped, narrowly funnel-shaped, boat-shaped, etc.
3.3 Polymorphism of ISSR markers
ISSR polymorphism analysis of Impatiens in eastern Yunnan revealed that 23 ISSR primers identified a total of 235 high allelic richness, 223 polymorphic bands, and a relatively high polymorphism ratio of 0.9490. Sites with a high polymorphism (66.7%). Primer UBC825 had the least polymorphic site, amplified 6 polymorphic bands, and the polymorphism ratio of 14 primers reached 100%, accounting for 60.9% of the total, with primers UBC848 and UBC861 amplifying up to 15 bands. And primers UBC848 and UBC811 could well distinguish 32 species of Impatiens in eastern Yunnan(The results are shown in Fig. 1). Therefore, the polymorphic loci in the genus Impatiens revealed the polymorphism difference of the genus,it indicated that the species richness of the genus Impatiens was higher in eastern Yunnan. (The results are shown in Table 2)
Cluster analysis was performed based on the genetic similarity coefficient of Impatiens in eastern Yunnan (Fig. 2), with GS values ranging from 0.540 to 0.990, it indicated significant genetic differences. The dendrogram was divided into three groups, with Group I divided into 4 subclades, SCI, SCII, SCIII, and SCIV. For SCI, accessions such as I. apsotis, I. longialata, and I. polyceras were all collected in Xundian County; I. siculifer var., unidentified species 2, I. racemosa, I. duclouxii, I. napoensis and I. napoensis var. were all collected in Pingbian County. I. duclouxii, I. napoensis and I. napoensis var. were tightly clustered together, indicating that the three Impatiens species were closely related, and its flowers were larger, with round standard petals, short-handled, two-lobed petals, wide funnel-shaped blades, and very similar flower shapes. However, I. Siculifer and I. siculifer var. were not closely clustered, indicating that the variation of ISSR polymorphisms was larger than that of I. napoensis and I. napoensis var.. In SCII, I. linearisepala was divided into a single group, and it has 4 sepals, indicating that the number of sepals was also an important taxonomic indicator of the genus in eastern Yunnan. Similarly, I. oxyuanthera in SCIII was grouped separately. In SCIV, unidentified species 1, I. dicentra, I. pinfanensis, I. arctosepala, and I. desmantha were all collected in Zhaotong, indicating the genetic relationship among the genus Impatiens in the same area closer. Group II consisted of the unidentified species 5 and I. uliginosa, indicating that they were closely related, which provided a certain basis for the identification of the unidentified species 5. Group III contained I. mengtzeana and I. aquatilis, and they were all pink flowers of the balsam genus. (The results are shown in Fig. 2)
3.4 atpB-rbcL sequence polymorphism marker
A dendrogram analysis using atpB-rbcL markers identified five major groups (Fig. 3), also highlighting the significant territorial characteristics of of Impatiens in eastern Yunnan. For group I, three sub-clades of SCI, SCII, and SCIII were detected, and most of them were collected in Pingbian County and Xundian County. In SCI, I. polyceras, I. rubrostriata, unidentified species 2, I. siculifer, I. siculifer var., I. dichroa, I. aquatilis, I. mengtzeana, and unidentified species 3 were all clustered together, indicating that these species of Impatiens plants were closely related.
Among them, I. siculifer and I. siculifer var. were closely clustered, which differed from the ISSR clustered results; unidentified species 3 was closely clustered with I. siculifer and I. mengtzeana respectively in the two markers, which made the identification of its species more difficult. However, I. aquatilis and I. mengtzeana were closely clustered in the evolutionary tree of ISSR and atpB-rbcL sequence markers. In SCII, I. Cyathiflora was grouped separately, indicating that it was far from relatives of the genus Impatiens in SCI. In SCIII, it consisted of I. racemosa, I. longialata, I. arctosepala, I. radiata, I. apsotis, and unidentified species 4 were clustered together; among them, I. racemosa and I. longialata were closely clustered, indicating that the atpB-rbcL clustered was more closely related than ISSR clustered; the remaining three were consistent with the ISSR clustered results and were all closely clustered. Group II was divided into 2 subclades, SCI and SCII. In SCI, I. uliginosa and unidentified species 5 were grouped, which was consistent with the ISSR results, indicating that they were closely related. Therefore, it was speculated that the unidentified species 5 was very likely to be a variant of I. uliginosa. In SCII, I. napoensis var., I. napoensis, I. duclouxii, I. dicentra, I. corchorifolia, unidentified species 1, I. blepharosepala, I. pinfanensis, I. vittata, I. oxyuanthera, I. desmantha, I. alpicola, and I.bodinieri were clustered together; and I. napoensis var., I. napoensis, I. duclouxii were closely clustered, which was consistent with the ISSR results, indicating that their kinship was closer, and the clustered results were more reliable. Group III, I. linearisepala was clustered individually. In conclusion, these two molecular markers have high reliability for the genetic diversity analysis of Impatiens in eastern Yunnan.
3.5 trnL-trnF sequence polymorphism marker
The dendrogram was mainly divided into 3 groups. For group I was divided into 2 subgroups, SCI and SCII. In SCI, I. cyathiflora, I. aquatilis, unidentified species 3, I. mengtzeana, I. dichroa, I. siculifer, I.siculifer var., I. polyceras, unidentified species 2, and I. rubrostriata were clustered together; in SCII, I. radiata, unidentified species 4, I. apsotis, I. arctosepala, I. racemosa and I. longialata were clustered together, which the clustered results were the same as that of atpB-rbcL sequence clustered. And I. polyceras, unidentified species 2, I. rubrostriata were closely clustered together. While I. cyathiflora was not clustered individually among the trnL-trnF sequence polymorphism markers. In group II, I. bodinieri, I. napoensis, I. napoensis var. and I. duclouxii, unidentified species 5, I. uliginosa, unidentified species 1, I. corchorifolia, I. dicentra, I. blepharosepala, I. alpicola, I. desmantha, I. vittata, I. pinfanensis, and I. oxyuanthera were gathered together. The 15 species of Impatiens and the 15 species of Impatiens marked by atpB-rbcL sequence were homogenized into a group, it indicated that the clustering results of these two markers were consistent, and the chloroplast genome sequences (atpB-rbcL, trnL-trnF) could be used for the evolutionary analysis of Impatiens in eastern Yunnan. And in the atpB-rbcL sequence marker, I. alpicola and I. bodinieri were closely clustered together, indicating that their kinship was closer and the evolutionary relationship was obvious; but in the trnL-trnF sequence marker, the evolutionary relationship of I. bodinieri, I. dicentra, I. blepharosepala, etc. was not obvious. It indicated that the atpB-rbcL sequence marker was more effective for the genetic diversity analysis of the genus Impatiens in eastern Yunnan. In group III, I. linearisepala was grouped individually. Among group II, I. napoensis, I. napoensis var. and I. duclouxii were closely clustered in the three markers, and the unidentified species 5 and I. uliginosa were also closely clustered in the three markers; it showed that ISSR and the chloroplast genome sequences had certain applicability to the evolution analysis of Impatiens in eastern Yunnan, but the evolutionary relationship of chloroplast genome sequence markers was not obvious, while the genetic distance of ISSR was relatively clear, the evolutionary relationship was more obvious. (The results are shown in Fig. 4. )
3.6 DEF sequence molecular marker
The dendrogram was mainly divided into 4 groups. Group I was divided into 4 subgroups, SCI, SCII, SCIII, and SCIV. In SCI, I. oxyuanthera, I. alpicola, I. pinfanensis, I. vittata were clustered together; and I. desmantha, I. bodinieri were closely clustered, which was consistent with the results of atpB-rbcL and ISSR sequence clustered, indicating that they were closely related; and I. blepharosepala, I. siculifer, I. arctosepala, unidentified species 3 were tightly clustered together, which was consistent with the ISSR sequence clustered results. In SCII, I. dicentra was individually clustered into a subgroup. In SCIII, I. corchorifolia and unidentified species 3 were tightly clustered together. In SCIV, I. uliginosa and unidentified species 5 were tightly clustered, and I. duclouxii, I. napoensis, and I. napoensis var. were closely clustered, which was consistent with the other three marker results, further illustrating the reliability of the clustered results. The DEF sequence marker indicated that the five species were closely related to each other, and it were consistent with the trnL-trnF sequence results. However, in the atpB-rbcL sequence and ISSR sequence markers, I. uliginosa, unidentified species 5, I. duclouxii, I. napoensis, and I. napoensis var. were clustered into different groups, reflecting the clustering differences between the different markers. In group II, I. mengtzeana and I. aquatilis were grouped separately in a group, and the results were consistent with the ISSR. Using atpB-rbcL sequence, I. mengtzeana, I. aquatilis, and unidentified species 3 were closely clustered together; and their evolutionary relationship of them in trnL-trnF marker was not obvious. These results indicated that the nuclear gene sequence markers were more obvious in the evolutionary relationship of Impatiens in eastern Yunnan. For group III was divided into 3 subgroups, SCI, SCII, and SCIII. In SCI, I. cyathiflora clustered separately into a subgroup. In SCII, I. radiata, unidentified species 4, I. longialata, I.apsotis, and I. racemosa were grouped. However, in the chloroplast genome sequence markers, I. arctosepala was also closely clustered with these five species, which was different from DEF and ISSR sequence markers. Although these five species of Impatiens were clustered into a group in ISSR sequence markers, their clustering results were distant and their genetic relationship was unclear. In SCIII, I. siculifer var., undetermined species 2, I. polyceras, and I. rubrostriata were clustered together; but the clusters of I. siculifer and I. siculifer var. were far apart, indicating greater changes in nuclear genes. In group SCIV, I. linearisepala singly gathered into a group. These results indicated that the DEF sequence marker can better explain the relationship among Impatiens in this area. (The results are shown in Fig. 5. )